Microvascular endothelial dysfunction precedes the development of vascular disease and portends future adverse vascular events. Endothelial dysfunction is readily identified by a characteristic loss of nitric oxide (NO) bioavailability, whch results from the dysregulation of a wide variety of proteins and molecular pathways. However, we lack important knowledge of how these proteins and molecules are coordinately regulated. MicroRNAs have been demonstrated to act as master regulators of physiological or disease processes by coordinately targeting multiple genes involved in the process. However, the role of microRNAs in endothelial dysfunction, especially in the context of diabetes, remains largely unexplored. We have obtained preliminary data suggesting a homeostatic level of vascular miR-29b is critical for the maintenance of normal microvascular NO bioavailability and endothelium-dependent vasodilation in humans and animal models. Conversely, in type 2 diabetes mellitus, a disease typified by reduced microvascular NO bioavailability and microvascular morbidity, miR-29b could restore NO bioavailability and endothelium- dependent vasodilation. MiR-29b's impact on NO bioavailability appears to arise from its coordinated effects on several genes that alter NO bioavailability at multiple levels of regulation. We propose in this application to investigate the novel role of miR-29b in microvascular endothelium- dependent vasodilation and NO bioavailability in health and in type 2 diabetes and identify the mechanisms mediating these effects of miR-29b. The proposed project will employ a highly translational approach that combines functional studies of a newly developed knockout rat model with studies of intact human vessels obtained from well-phenotyped volunteers. We will also employ newly developed methods for identifying target genes and molecular pathways involved in the effect of miR-29b. Specifically, we will test the hypothesis that endogenous miR-29b is critical to maintaining normal endothelium-dependent vasodilation and NO bioavailability in resistance vessels in healthy humans and animals in Aim 1. Aim 2 studies will test the hypothesis that miR-29b can restore endothelium-dependent vasodilation and NO bioavailability in resistance vessels from T2DM patients and db/db mice. The molecular mechanisms underlying the role of miR-29b in endothelium-dependent vasodilation and NO bioavailability will be examined in Aim 3. The study will be carried out by a team of experienced researchers led by a physician scientist and a basic scientist who possess complementary expertise ideally suited for the proposed project. Successful completion of the project will reveal novel mechanisms regulating endothelial function and demonstrate their clinical relevance.